Project Summary Constitutively increased intracellular pH (pHi) is common to most cancers regardless of tissue origin or genetic background. Increased pHi is necessary for survival of patient-derived tumor cells, and is sufficient to induce oncogenic phenotypes, including dysregulated tissue growth, dysplasia, and invasive cell migration. pH-regulated cell behaviors are predominantly mediated by changes in the protonation state of pH sensitive proteins, or pH sensors, which is viewed as a post-translational modification which cannot be resolved by mass spectrometry or antibodies. At the molecular level, changes in pHi can alter the protonation state of selective amino acid residues with pKa values near neutral, which can markedly affect protein conformations and function. By changing the protonation of multiple proteins in unison, pHi dynamics can coordinate complex cell processes. Although understudied, emerging evidence suggests that the higher pHi of most cancers contributes to pathogenesis. In this proposal, we will characterize pH sensitive cancer cell behaviors and identify pH sensors that regulate proliferation (Aim 1), programmed cell death (Aim 2) and metastasis (Aim 3). Using the genetic model organism Drosophila, we will bridge scales from tissue-level phenotypic analyses to individual pH-sensitive proteins that regulate these cancer cell behaviors. We will use cutting edge technologies including fluorescent cell cycle and apoptosis markers, genetically encoded pH bio-sensors, and quantitative imaging techniques to generate 3-D maps of intratumoral pHi. Our unpublished data with genetic modifier screens identified a number of candidate pH sensors with known roles in regulating these processes. We will test predictions on the pH-sensitive function of these candidate pH sensors. Significant outcomes from our studies include new insights on how pH-regulated cellular behaviors enable tumorigenesis and metastasis. The strengths of the PI in linking tissue-level phenotypes to individual pH-sensitive molecules permit unique insights in this under-studied area of biology. Further, understanding the molecular mechanisms of pH-sensitive proteins will inform novel therapeutic approaches to limit cancer progression.